Methods of using BCL-2 for the therapeutic treatment and prevention of diseases

ABSTRACT

The invention provides a method of treating a disease or pathological condition resulting in apoptotic cell death. The method includes increasing the activity of Bcl-2 in cells affected by the disease or pathological condition. Diseases or pathological conditions can include, for example, neurodegenerative diseases, cancer and viral infections. Also provided is a method of prolonging the in vivo survival of transplanted cells for the treatment of a disease or pathological condition. The method includes increasing the activity of Bcl-2 in a population of cells and transplanting the population of cells having increased Bcl-2 activity into a subject. Diseases or pathological conditions can include, for example, neurodegenerative diseases, cancer and viral infections. A method to enhance the sensitivity of malignant cells to therapy is provided that includes decreasing the activity of Bcl-2 in the malignant cells. Methods to identify compounds that alter apoptotic cell death and to enhance monoclonal antibody production are also provided by the invention disclosed herein.

This application is a continuation of application Ser. No. 10/285,853,filed Nov. 1, 2002, now U.S. Pat. No. 7,427,495, which is a continuationof application Ser. No. 08/625,761, filed Mar. 29, 1996, now abandoned,which is a divisional of application Ser. No. 08/066,556, filed May 26,1993, now U.S. Pat. No. 5,550,019, each of which is incorporated hereinby reference in its entirety.

This invention was funded in part by NIH Grant CA-47956. Accordingly,the United States government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Apoptosis is the term used to describe a type of cellular death thatoccurs in many tissues as a normal physiological process. Also called“programmed cell death,” this form of cellular demise involves theactivation in cells of a built-in genetic program for cell suicide bywhich cells essentially autodigest. The remnants of these dead cells arethen cleared almost without a trace by neighboring phagocytic cells,without resulting in inflammation or scarring. Apoptosis thus stands inmarked contrast to cell death caused, for example, by oxygen-deprivationin the settings of myocardial infarction or stroke, where cells losetheir energy supplies, rupture and spill their contents into theextracellular milieu.

In addition to the normal physiological process where cells are turnedover within the body, apoptosis can be induced to occur by cellular,hormonal or other stimuli to remove unwanted cells from the body. Forexample, killing of tumor cells and virus-infected cells by the immunesystem's cytolytic T-cells occurs via apoptosis following targetrecognition. Apoptosis also occurs via loss of hormonal stimulation inthe female reproductive tissues with each menstrual cycle in the absenceof a successful pregnancy. Further, numerous studies have shown thatapoptosis accounts for cell death in a wide variety of clinicallyimportant areas. For example, essentially all chemotherapeutic drugscurrently used in the treatment of cancer, as well as x-irradiation inmany cases, ultimately kill malignant cells by activating intracellularpathways leading to apoptosis.

In contrast to the effect of apoptosis in normal cellular phenomenon,when aberrantly regulated, the death of cells through apoptosis can leadto a variety of disease states and pathological conditions. For example,the death of neurons that occurs in diseases such as Alzheimer'sdementia and Parkinson's disease shows many hallmarks of apoptosis.Autoimmune diseases, where immune cells inappropriately attack normaltissues, is due, in part, to a failure of apoptosis to occur.Additionally, cell death caused by viral infection can occur throughapoptosis in many cases, including T-cell death induced by the HumanImmunodeficiency Virus (HIV) that causes AIDS. In contrast to theinduction of apoptosis caused by some viruses, other viruses inhibitthis process through the expression of gene products that blockapoptosis. Herpes Simplex virus is a specific example of this inhibitionwhere the prevention of apoptosis is necessary for its characteristicpersistent or “latent” viral infection.

Efforts have been made using conventional chemotherapy to treat many ofthe disease states that result in inappropriate apoptotic cell death,including all of those mentioned above, but have so far yielded onlyminor progress toward an effective treatment. Additionally, othernon-conventional approaches have also been tried in specific enhances.For example, Parkinson's disease has been treated in humans using fetalneural tissue transplantation. Extensive testing of such neural tissuetransplants as well as testing of genetically engineered fibroblasts hasbeen investigated in animal models of Parkinson's and Alzheimer'sdisease. In the latter case, fibroblasts were modified to secreteneurotrophic factors such as nerve growth factor (NGF) orneurotransmitters such as precursors of dopamine to prolong recipientneural cell survival. The general difficulty of these treatments is thateither only a small number of the transplanted cells initially surviveupon implantation, or in the case of genetically-engineered fibroblasts,many of the cells that initially survive fail to continue long-term invivo survival.

As mentioned previously, cancer chemotherapy acts through a variety ofintracellular targets which culminate in the activation of the apoptoticpathway. Cancer is the second leading cause of death in the UnitedStates. One out of every 3 Americans will develop some form of thisdisease in his or her lifetime, and the vast majority will die as adirect result of their malignancies, primarily because of the inadequacyof currently available chemotherapeutic drugs or the spontaneousresistance of malignant cells to such treatments.

Although many details of malignancies are not fully understood, thebasis of a variety of cancers have been worked out in large part. Forexample, the unregulated expression of many genes is now known to causeoncogenic transformation. These genes have generically been termedoncogenes because of their transformation ability when abnormallyexpressed. Typical examples of such oncogenes include protein kinasessuch as Src, Herb-2 (NEU), and BCR/ABL, GTPases such as Ki-RAS, Ha-RAS,and N-RAS, and transcription factors such as Fos, Jun and Myc. These andother oncogenes have been well documented to be a major cause in themalignant phenotype of cancers such as neuroblastoma (N-myc), Burkittlymphoma (c-myc), colorectal carcinoma (Ki-RAS), chronic myelogenousleukemia (BCR/ABL), breast cancer (Herb-2/NEU), and lung cancer (L-myc).Attempts have been made to target several of these oncogenes to providea therapeutic treatment for the relevant cancer. However, because of theproblems mentioned above in regard to chemotherapy, such attempts alltoo often have proven to be of marginal benefit.

In contrast to oncogenes whose abnormal or unregulated expressionresults in increased proliferation, there is at least one “oncogene”whose overexpression results in prolonged cell survival. This oncogeneis termed Bcl-2 and was originally discovered because of itsinappropriate activation in lymphomas, cancers of the lymph nodes, whereit contributes to neoplastic cell expansion. High levels of Bcl-2protein have been demonstrated to occur in approximately 50,000 newcases of lymphoma and leukemia each year in the United States alone,essentially all cases of drug-resistant prostate cancer (150,000 casesper year in USA), 80% of nasopharyngeal carcinomas, about 70% of breastcancers (approximately 100,000 cases per year in the United States) andall cases of colorectal carcinoma examined to date (110,000 new casesper year in USA).

Since its initial discovery, it has been shown that Bcl-2 is alsonormally expressed in many tissues of the body, where it serves aphysiological role of maintaining the survival of long-lived cells.Among the types of cells whose survival Bcl-2 regulates are the “memory”lymphocytes that are generated during immunizations, many types ofneurons in the brain and particularly in the peripheral nerves thatcontrol muscle and organ functions, bone marrow cells, skin, and thestem cells giving rise to the absorptive cells that line thegastrointestinal tract.

From the foregoing discussion, it is apparent that for many of thediseases that impact the human species, there have been no majoradvances toward an effective treatment within the last 10 to 15 years.However, as diverse as these diseases and their treatments are, therehas developed one common mechanism by which they manifest theircharacteristics or by which chemotherapy has been rendered ineffectual.This common mechanism is either the induction or inhibition ofprogrammed cell death.

Thus, there exists a need to control the process of apoptosis in orderto generically treat a broad range of diseases and pathologicalconditions, and also to be able to augment clinical treatments employingreadily available drugs. The present invention satisfies this need andprovides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides a method of treating a disease or pathologicalcondition resulting in apoptotic cell death. The method includesincreasing the activity of Bcl-2 in cells affected by the disease orpathological condition. Diseases or pathological conditions can include,for example, neurodegenerative diseases, cancer and virus infectedcells. Also provided is a method of prolonging the in vivo survival oftransplanted cells for the treatment of a disease or pathologicalcondition. The method includes increasing the activity of Bcl-2 in apopulation of cells and transplanting the population of cells havingincreased Bcl-2 activity into a subject. Diseases or pathologicalconditions can include, for example, neurodegenerative diseases, cancerand virus infected cells. A method to enhance the sensitivity ofmalignant or virus infected cells to therapy is provided that includesdecreasing the activity of Bcl-2 in the malignant or virus infectedcells. Methods to identify compounds that alter apoptotic cell death andto enhance monoclonal antibody production are also provided by theinvention disclosed herein as well as by using transgenic miceexpressing Bcl-2 as the transgene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Recombinant Bcl-2 retroviral vectors constructed using standardrecombinant DNA techniques.

A. Cloned human DNA sequences encoding either the full length Bcl-2protein (“bcl-2α”) or a shorter inhibitory form of Bcl-2 (“bcl-2β”) wereinserted into the unique XhoI site in pBC140 in both the forward andreverse (AS; antisense) orientations. Bcl-2 expression is driven by acytomegalovirus promoter/enhancer (CMV; black bars). Neomycinphosphotransferase (Neo^(r)) gene expression was driven by the left-mostMoloney virus long terminal repeat (LTR; white bars) and used as adominant selectable marker using the antibiotic G418. Selectedrestriction sites are indicated.

B. A human Bcl-2 cDNA encoding the full length Bcl-2 protein was clonedinto the unique BamHI site in pZip-NEO. Selected restriction sites areindicated: X, XhoI; Xb, XbaI; B, BamHI. Bcl-2 expression driven by theleft-most Moloney virus LTR. Expression of the neomycin resistance geneis mediated via a splicing event (not shown).

FIG. 2. Bcl-2 and a mutant Bcl-2 (59 Pro->Ser) protein allow mammaliancells to grow to higher cell densities.

A. Cell viability as determined by trypan blue exclusion (mean +/−standard deviation of 3 determinations). W.T., wild-type Bcl-2; P59S,mutant Bcl-2; Neo, control.

B. DNA synthesis as determined by liquid scintillation counting of cellspulse-labeled for 8 hours with ³H-thymidine. Kcpm=kilocounts/min (mean+/− standard deviation of 3 determinations).

FIG. 3. Bcl-2 blocks prostate cancer cell line, AT-3, death induced bySindbis virus infection. Cell viability as determine by trypan blueexclusion (mean +/− standard deviation of 3 determinations). pZIP-BCL2or pZIP-NEO are described in FIG. 1.B. AT-3-NEO control cells (circles);AT-3-BCL2 cells (squares). Virus infected cells (solid symbols); cellscultured in the absence of virus (open symbols).

FIG. 4. Protection of PC12 cells from glutamate-induced death by Bcl-2.Cell viability as determined by MTT dye reduction assay (mean +/−standard deviation of 3 determinations).

FIG. 5. Homologs of Bcl-2 prolong survival of 32D-3 cells grown in theabsence of Il-3 for various times. Cell viability as determined by thetrypan blue exclusion (mean +/− standard deviation of 3 determinations).

A. Human (HU) and chicken (CH) Bcl-2 proteins prolong cell survival.(NEO=control; no Bcl-2).

B. Human Bcl-2 and the Epstein Barr virus Bcl-2 homolog, BHRF-1, prolongcell survival. (HYG=control; hygromycin B).

FIG. 6. Bcl-2 acts synergistically with Raf-1 to prolong cell survivalunder conditions of growth factor deprivation. Bcl-2 protein, alone(open circles); activated Raf-1 protein, alone (squares); Bcl-2+Raf-1proteins (closed circles); no Bcl-2 or Raf-1 protein (triangles).

A. Cell viability as determined by trypan blue exclusion (mean +/−standard deviation of 3 determinations).

B. DNA synthesis as determined by 3H-thymidine incorporation. Data werenormalized by dividing the results obtained for cells cultured in theabsence of interleukin-3 (Il-3) by values obtained for cells cultured inthe presence of Il-3 (mean +/− standard deviation of 3 determinations).

C. Relative DNA content of propidium-iodide-stained viable cells asdetermined by flow cytometry. Cells were grown for 3 days in thepresence (panels a and c) or absence (panels b and d) of Il-3. 32D-BCL2cells (panels a and b); 32D-BCL2/RAF cells (panels c and d).(FL=fluorescence intensity).

FIG. 7. A dominant-negative form of the Bcl-2 protein, Bcl-2β,accelerates cell death. Cell viability as determined by trypan blueexclusion. Data were normalized relative to the control cells (NEO)(mean +/− standard deviation of 4 determinations).

FIG. 8. Western blot of Sf9 cell extracts. Lanes 1-3 contain 5.0, 1.0 or0.2 μg, respectively, protein from recombinant Bcl-2baculovirus-infected cells; lane 4, 100 μg protein fromt(14;18)-containing human B cell lymphoma cell line; lane 5, 10 μgprotein from recombinant Lck baculovirus-infected cells (negativecontrol); lane 6, 10 μg protein from uninfected cells.

FIG. 9. Cell-free assay for assessing Bcl-2 function.

A. Nuclei incubated in cell free extracts containing Sf9 insect celllysates prepared from control (upper panels) or Bcl-2-expressing (lowerpanels) Sf9 cells. Nuclei visualized using Hoechst 33258 DNA stain(DNA); tetramethylrhodamine-labelled BSA conjugated to a syntheticpeptide corresponding to the nuclear localization signal of SV40T-antigen (TRITC-HSA-NLS); phase contrast microscopy (PHASE CONTRAST).

B. Quantitation of intact, transport-competent nuclei incubated for 120or 160 min in the presence of absence of Bcl-2.

FIG. 10. Bcl-2 protein is directly responsible for suppression ofnuclear breakdown in cell-free apoptosis assays.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to general and effective methods to augmentthe treatment of diseases and pathological conditions. The methods areapplicable to the treatment of cancer, neurodegenerative disorders,viral infections, autoimmune diseases and also to the modification oftransplanted tissues and cells. The modified tissues and cells can beused, for example, in the treatment of neurological disorders, as wellas diseases caused by hormonal and protein insufficiencies such asdiabetes (insulin) and hemophilia (coagulation factors). The methodsdescribed herein also enable the development of novel pharmaceutics forthe treatment and prevention of diseases and pathological conditions.Additionally, the methods of this invention can be further applied tothe production of superior research and diagnostic reagents andcompositions for use in essentially all disciplines of the basic andapplied sciences.

The invention takes advantages of the ability of Bcl-2 to prevent theprocess of programmed cell death known as apoptosis. Targeting aphysiological mechanism common to many diverse diseases is efficient andcost effective in that the specialized development of different drugs toeach of the specific diseases is not required. Instead, a small numberof therapeutic compounds or methods of treatment can be developed forthe treatment of essentially all diseases that manifest theirpathological condition through the aberrant regulation of apoptosis. Inmany cases, such compounds can be recombinant nucleic acids whoseadministration is by mode of gene therapy. Thus, reagents that eitherpromote or impair apoptosis are used in the methods of the invention toretard disease-induced apoptotic cell death or to selectively enhancetumor cell killing by conventional chemotherapeutic drugs as well as toprotect normal non-neoplastic cells from the toxicity of these drugs.

In one embodiment, gene transfer technology or “gene therapy” is used tocreate Bcl-2 recombinant DNA molecules and viral vectors to promote thein vivo survival of cells affected by a disease or pathologicalcondition that results in apoptotic cell death. Bcl-2 recombinant DNAmolecules and viral vectors are also used to genetically modify cellsprior to transplantation to prolong their in vivo survival time and,where applicable, to simultaneously correct protein deficiencies. Theuse of Bcl-2 to immortalize or prolong the survival rate of targetedcells is advantageous in that it does not block cellular differentiationand is essentially non-tumorigenic when expressed in either primary orestablished cells compared to other oncogenes. Specific examples of suchtherapies include the production of recombinant viruses that directBcl-2 expression to specific types of neurons and the administrationsuch viruses to patients having Alzheimer's or Parkinson's diseases viadirect injection into the brain or spinal fluid; the intracranialimplantation of Bcl-2 expressing fetal neuronal precursor cells for thetreatment of Parkinson's disease; the mass expansion of human Bcl-2expressing cells in vitro for prolonging their in vivo survival aftertransplantation and the use of recombinant vectors for directing Bcl-2expression to specific cell types for the prevention of cell deathinduced by viral infections. Bcl-2 expressing cells for use intransplantations can be genetically modified, for example, to secretevarious hormones and peptides such as neurotrophic factors in thesetting of spinal cord injury, dopamine for the treatment of Parkinson'sdisease, enkephalins for pain control in terminally ill cancer patients,coagulation factors for patients with hemophilia and insulin producingislet cells for patients with diabetes.

In another embodiment, Bcl-2 gene transfer technology is used to“immortalize” human antibody-producing B-cells and thereby enhance thedevelopment of human monoclonal antibodies. The survival promotingfunction of Bcl-2 is also utilized to generate Bcl-2 transgenic mice forisolating B-cells for enhanced monoclonal antibody production. Suchmonoclonal antibodies are useful for diagnostic and therapeuticpurposes.

In yet another embodiment, a cell-free system is described thatfaithfully reproduces characteristics of apoptotic cell death. Thesystem is useful for the screening of compounds that alter the apoptoticprocess. Selected compounds that either promote or inhibit apoptosis canbe used for therapeutic treatment of a variety of diseases includingneurodegenerative diseases, cancer and virus infected cells.

The Bcl-2 gene was first discovered because of its-involvement inlymphomas in humans. This gene has now been shown to prolong cellsurvival in culture when expressed at high levels in a variety of celltypes such as lymphocytes, hemopoietic cells, fibroblasts and neurons.Bcl-2 is a 26 kilodalton (kDa) protein that is unique among cellulargenes. It contains a stretch of 17 hydrophobic amino acids near itscarboxy terminus that causes its post-translational insertion intointracellular membranes. Gene transfer studies have demonstrated thatBcl-2 promotes cell survival by blocking programmed cell death, orapoptosis.

Apoptosis can be actively triggered in cells by, for example, exposureto X-radiation, cytotoxic drugs, free-radicals and heat, or it can beunmasked by removal of critical peptide growth factors, steroidhormones, lymphokines or neurotrophins that constantly suppressprogrammed cell death in various tissues. Many of these processes arethe terminal events involved in numerous disease states or the finalevents by which therapeutic treatments effect their results. Thus, tospecifically target and alter apoptosis would provide a generaltreatment for a broad range of diseases and pathological conditions. Itshould be noted, however, that there exist Bcl-2-independent pathwaysfor apoptosis. Thus, the new uses for Bcl-2 reported here constitutepreviously undocumented circumstances under which Bcl-2 gene transfer isrevealed for the first time to exert protection against programmed celldeath.

Bcl-2 is normally expressed in a variety of types of cells butparticularly those that either exhibit a long lifespan such as sometypes of neurons, long-lived “memory” lymphocytes or cell havingproliferative, self-renewing potential such as basal epithelial cellsand hemopoietic progenitor cells in the bone marrow. It is likely thatBcl-2 represents the prototype of an entire family of structurallysimilar genes that are expressed in a tissue-specific manner andcontribute to the regulation of cellular life span.

As used herein, the term “apoptosis” or “apoptotic cell death” refers tothe physiological process known as programmed cell death. Apoptosis isunlike other forms of cell death that occur, for example, as the resultof ischemia or necrosis because apoptosis is an active, ATP-requiringform of cell death that typically requires new RNA and proteinsynthesis. A hallmark of apoptosis is the activation of endogenousendonucleases that initially cleave the genomic DNA at its mostaccessible sites, i.e., between nucleosomes, producing a ladder of DNAbands representing integer multiples of the internucleosomal distance.This DNA degradation occurs early in the apoptotic process, before lossof plasma membrane integrity. Apoptotic cells also have a shrunken sizeand the process is not usually accompanied by inflammation since thereis no spilling of cytoplasmic contents into the extracellular space.With apoptosis, much of the cell's content is autodigested. In vivo celllysis never occurs because the apoptotic cells are usually phagocytosedby macrophages and related cells before loss of plasma membranepermeability. Consequently, there is no inflammatory reaction orsubsequent scarring. Other morphological characteristics of apoptoticcells include nuclear fragmentation, development of vesicular bodies,“apoptotic bodies” and plasma membrane blebbing, all in the setting ofintact mitochondria and lysosomes. Specific examples of apoptotic celldeath as a natural programmed event include, for example, the loss ofredundant neurons during fetal development and the destruction ofpotentially autoreactive T-cells during thymic education.

As used herein, the term “Bcl-2” refers to the protein originallydiscovered due to its inappropriate activation in lymphomas. Bcl-2controls normal cell growth and differentiation by promoting cellsurvival. It has a molecular weight of about 26 kDa as determined bySDS-PAGE and is characterized by a hydrophobic stretch of about 17 aminoacids near its carboxy terminus that functions in intracellular membraneattachment.

Bcl-2 has substantially the same amino acid sequence as that shown inGenBank accession M13994 and is encoded by a nucleotide sequencesubstantially similar to that shown as GenBank accession M13994. Thedefinition of “Bcl-2” is intended to include other Bcl-2 family memberssuch as those proteins that are found to exhibit the above functionalcharacteristic or sequence homologies. Such members include, forexample, homologs of Bcl-2 cloned from lower organisms such as rats,mice, chickens, flies and worms.

A specific example of a Bcl-2 family member is the protein encoded bythe BHRF-1 gene in Epstein Barr virus. The BHRF-1 gene, which exhibitsabout 22% sequence identity and 47% sequence similarity with Bcl-2, isfunctionally equivalent to Bcl-2 in promoting cell survival (see, forexample, FIG. 5).

It is understood that limited modifications to the protein can be madewithout destroying the biological function of Bcl-2 and that only aportion of the entire primary structure may be required in order toeffect activity. For example, minor modifications of the Bcl-2 proteinor nucleotide sequence which do not destroy its activity are includedwithin the definition of Bcl-2. Moreover, fragments of Bcl-2 whichretain at least one function of the entire protein are included withinthe definition. It is understood that various modifications of primaryamino acid or nucleotide sequence may result in proteins havingsubstantially equivalent or enhanced function as compared to thesequences set forth in GenBank accession number M13994. Thesemodifications may be deliberate, as through site-directed mutagenesis,or accidental, such as through mutation in hosts which are Bcl-2producers. All of these modifications are included as long as Bcl-2biological function is retained. Furthermore, various molecules, such asother proteins, carbohydrates, or lipids, can be attached to Bcl-2. Suchmodifications are included within the definition of Bcl-2.

The invention provides a method of treating a disease or pathologicalcondition resulting in apoptotic cell death. The method includesincreasing the activity of Bcl-2 in cells affected by the disease orpathological condition. Diseases or pathological conditions can include,for example, neurodegenerative diseases, cancer and virus-infectedcells.

Alzheimer's disease, the most common neurodeqenerative disorder, isestimated to affect four million Americans and represents a majoreconomic burden to families and society. No treatment can stop or evenslow the progression of this disorder. Amyloid β-protein (ABP) has beenidentified as a possible causative agent of this disease. Addition ofABP, or of specific peptide fragments from this protein, to culturedneurons and neuronal cell lines results in cell death. Expression ofBcl-2 in these cultured cells by gene transfer can reduce neuronal cellkilling by ABP. These results indicate that apoptosis contributes toneuronal cell death in Alzheimer's disease.

Parkinson's disease is a progressive and ultimately fatalneurodeqenerative disorder characterized by loss of the pigmenteddopaminergic neurons of the substantia nigra. The symptoms ofParkinson's disease can often be managed initially by administration ofL-DOPA, the immediate precursor of dopamine. However, reduced efficacyof L-DOPA treatment often occurs possibly because metabolism of the drugprevents effective delivery to the CNS. Programmed cell death has alsobeen implicated to play an important role in this neurodegenerativedisorder inasmuch as withdrawal of neurotrophic factors from neuronsleads to cell death through a mechanism consistent with apoptosis.Moreover, the absence of inflammatory cells or scar formation in thebrains of patients with Parkinson's disease indicates that striatalneuron death can occur through apoptosis as opposed, for example, tonecrosis.

In addition to neurodegenerative disorders, apoptosis has been indicatedto result in cell death from glutamate-induced neurotoxicity arisingfrom conditions such as stroke and amyotrophic lateral sclerosis (ALS;“Lou Gehrig's disease”). Glutamate-induced toxicity occurs whenglutamate is released from dying neurons in the brain at times of acuteinjury. Glutamate released by dying neurons in turn binds to specificreceptors for glutamate on adjacent healthy neurons, triggering signalsthat set-off a complex series of biochemical events leading to apoptoticcell death.

Diseases and pathological conditions such as those described above andthose that will be described below can be treated by increasing theactivity of Bcl-2 in the cells affected by the disease or pathologicalcondition. Increasing Bcl-2 activity in these affected cells willinhibit the apoptotic death of such cells and therefore reduce orprevent progression of the disease or pathological condition.

The activity of Bcl-2 can be increased by a variety of means, including,for example, increasing the Bcl-2 synthesis rate or decreasing the Bcl-2degradation rate or modulating the ability of Bcl-2 to interact withother proteins that control the apoptosis process. Increasing thesynthesis rate of Bcl-2 will result in elevated protein accumulation andthereby increase Bcl-2 activity within the cell.

An elevated synthesis rate can be achieved, for example, by usingrecombinant expression vectors and gene transfer technology to express aBcl-2-encoding nucleic acid. Such methods are well known in the art andare described below with reference to recombinant viral vectors. Othervectors compatible with the appropriate targeted cell can accomplish thesame goal and, therefore, can be substituted for recombinant viralvectors in the methods described herein. For example, recombinantadenoviruses having general or tissue-specific promoters can be used todrive Bcl-2 cDNA expression and to deliver Bcl-2 expression constructsinto a variety of types of tissues and cells, including non-mitoticcells such as neurons in the substantia nigra of the brain (the regionaffected in Parkinson's disease) (La Salle et al., Science 259:988-990(1993), which is incorporated herein by reference).

Alternatively, recombinant adeno-associated viruses can be used for thispurpose, with the added advantage that the recombinant virus can stablyintegrate into the chromatin of even quiescent non-proliferating cellssuch as neurons of the central and peripheral nervous systems (Lebkowskiet al., Mol. Cell. Biol. 8:3988-3996 (1988), which is incorporatedherein by reference). Receptor-mediated DNA delivery approaches also canbe used to deliver Bcl-2 expression plasmids into cells in atissue-specific fashion using a tissue-specific ligand or antibodynon-covalently complexed with DNA via bridging molecules (Curiel et al.,Hum. Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol. Chem.262:4429-4432 (1987), both of which are incorporated herein byreference). Direct injection of DNA (mammalian expression plasmids ofvarious types) or of DNA encapsulated in cationic liposomes also can beused for stable gene transfer to non-dividing and dividing cells in vivo(Ulmer et al., Science 259:1745-1748 (1993), which is incorporatedherein by reference). In addition, DNA transfer by the particlebombardment method can be used to transfer DNA into a variety of tissues(Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991), whichis incorporated herein by reference).

Moreover, recombinant expression vectors encoding Bcl-2 can also containadditional non-Bcl-2-encoding nucleic acids that are useful for thetherapeutic treatment of a disease or pathological condition. Forexample, Bcl-2-encoding vectors can be constructed to encode enzymesused in the synthesis of dopamine when treating Parkinson's disease, forexample, with the idea of simultaneously providing genes for enhancementof cell survival (Bcl-2) and cell function (dopamine-β-hydroxylase).Other examples are Bcl-2 plus recombinant DNA sequences engineered toallow for nerve growth factor secretion for sustaining the survival ofthe cholinergic neurons that are typically lost in Alzheimer's disease(Rosenberg et al., Science 242:1575-1578 (1988), which is incorporatedherein by reference), Bcl-2 plus insulin for the treatment of diabetesor Bcl-2 plus encephalin for treatment of intractable pain. Whetherother non-Bcl-2-encoding nucleic acids are also contained within avector will depend on the disease and the therapeutic need. One skilledin the art will be able to determine such a need.

Viruses are very specialized infectious agents that have evolved in manycases to elude host defense mechanisms. Typically, viruses infect andpropagate in specific cell types. The targeting specificity of viralvectors utilizes this natural specificity, in turn, to specificallytarget predetermined cell types and, thereby, introduce a recombinantgene engineered into the viral genome into the infected cell. The vectorto be used in the methods of the invention will depend on desired celltype to be targeted. For example, if neurodegenerative diseases are tobe treated by increasing the Bcl-2 activity of neuronal cells affectedby the disease, then a vector specific for cells of the neuronal celllinage could be used. Such viral vectors include, for example, Herpessimplex virus-based vectors (Battleman et al., J. Neurosci. 13:941-951(1993), which is incorporated herein by reference). Similarly, if adisease or pathological condition of the hematopoietic system is to betreated, then a viral vector that is specific for blood cells and theirprecursors, preferably for the specific type of hematopoietic cell,should be used. Such viral vectors include, for example, HIV-basedvectors (Carroll et al., J. Cell. Biochem. 17E:241 (1993), which isincorporated herein by reference).

Moreover, such vectors can additionally be modified with specificreceptors or ligands to modify or alter target specificity throughreceptor mediated events. These modification procedures can beperformed, for example, using recombinant DNA techniques or syntheticchemistry procedures. Specific examples of viral vectors and theirspecificity include, for example, Herpes simplex virus for neuronal celllineages, HIV for T lymphocytes, hepatitis virus for liver cells andAdenovirus for lung and other tissues. In cases where viral infectionscannot be made tissue-specific, it may be possible to make viral geneexpression specific for only the desired type of cell through the use oftissue-specific promoters and enhancers (Dai et al., Proc. Natl. Acad.Sci. USA 89:10892-10895 (1992), which is incorporated herein byreference).

Viral vectors commonly used for in vivo targeting and therapy proceduresare retroviral vectors or DNA-based vectors. Retroviral vectors can beconstructed to function either as infectious particles or to undergoonly a single initial round of infection. In the former case, the genomeof the virus is modified so that it maintains all the necessary genes,regulatory sequences and packaging signals to synthesize new viralproteins and RNA. However, oncogenic transformation properties of theseviruses are destroyed. Once the viral proteins are synthesized, the hostcell packages the RNA into new viral particles, which can undergofurther rounds of infection. The viral genome is also engineered toencode and express the desired recombinant gene.

In the case of non-infectious viral vectors, the helper virus genome isusually mutated to destroy the viral packaging signal, which is requiredto encapsulate the RNA into viral particles, but retains the structuralgenes required to package the co-introduced recombinant-virus containinga gene or genes of interest. Without such a signal, any particles thatare formed will not contain a genome and, therefore, cannot proceedthrough subsequent rounds of infection.

The methods for constructing and using such viral vectors are known inthe art and are reviewed, for example, in Miller and Rosman,Biotechniques 7:980-990 (1992), which is incorporated herein byreference. The specific type of vector will depend upon the intendedapplication. The actual vectors are also known and readily availablewithin the art or can be constructed by one skilled in the art.

Bcl-2-encoding viral vectors can be administered in several ways toobtain expression and, therefore, increased activity of Bcl-2 in thecells affected by the disease or pathological condition. If viralvectors, for example, are used, the procedure can take advantage oftheir target specificity and the vectors need not be administeredlocally at the diseased site. However, local administration can providea quicker, more effective treatment. Administration can also be byintravenous or subcutaneous injection into the subject. Injection of theviral vectors into the spinal fluid can also be used as a mode ofadministration, especially in the case of neurodegenerative diseases.Following injection, the viral vectors will circulate until theyrecognize host cells with the appropriate target specificity forinfection.

An alternate mode of administration of Bcl-2 encoding vectors can be bydirect inoculation locally at the site of the disease or pathologicalcondition. Local administration is advantageous because there is nodilution effect and, therefore, a smaller dose is required to achieveBcl-2 expression in a majority of the targeted cells. Additionally,local inoculation can alleviate the targeting requirement needed withother forms of administration since a vector can be used that infectsall cells in the inoculated area. If expression is desired in only aspecific subset of cells within the inoculated area, then promoter andexpression elements that are specific for the desired subset can be usedto accomplish this goal. Such non-targeting vectors can be, for example,viral vectors, viral genomes, plasmids, phagemids and the like.Transfection vehicles such as liposomes can be used to introduce thenon-viral vectors described above into recipient cells within theinoculated area. Such transfection vehicles are known to one skilledwithin the art.

The Bcl-2 encoding nucleic acid used in the methods of the invention isknown in the art and available from Genbank under the locusidentification “HUMBCL2A” as accession number M13994. The nucleotidesequence available from the above database is all that is necessary forone skilled in the art to obtain a Bcl-2 cDNA for use in the disclosedmethods. Moreover, since the Bcl-2 cDNA has been published in Tsujimotoand Croce, Proc. Natl. Acad. Sci. USA, 83:5214-5218 (1986), which isincorporated herein by reference, it is also readily available to thoseskilled in the art.

Using the Bcl-2 sequence described in GenBank accession number M13994,one skilled in the art can clone the Bcl-2 using conventional libraryscreening methods. Oligonucleotide probes useful for screening can besynthesized using known methods in the art such as phosphoramiditechemistry. Other methods such as the polymerase chain reaction (PCR)also can be used to rapidly and efficiently clone Bcl-2-encoding nucleicacids. Using PCR, a DNA segment of up to approximately 6,000 base pairsin length can be amplified from a single gene copy.

Briefly, PCR involves incubating a denatured DNA sample with twooligonucleotide primers that direct the DNA polymerase-dependentsynthesis of complementary strands. Multiple cycles of synthesis areperformed, wherein each cycle affords an approximate doubling of theamount of target sequence. Each cycle is controlled by varying thetemperature to permit denaturation of the DNA strands, annealing theprimers and synthesis of new DNA strands. Use of a thermostable DNApolymerase eliminates the necessity of adding new enzyme for each cycleand permits fully automated DNA amplification. Twenty-five amplificationcycles increase the amount of target sequence by approximately 10⁶-fold.The PCR technology is the subject matter of U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065, and 4,683,202 all of which are incorporated hereinby reference.

The invention also provides a method of treating a disease orpathological condition resulting in apoptotic cell death by increasingthe activity of Bcl-2 wherein the disease or pathological condition ismediated by viral infection. The methods described above for thetreatment of neurological diseases and pathological conditions can alsobe applied to various other disease states such as virus-infected cells.Many viral infections, such as HIV, culminate in cell death throughapoptosis.

Apoptosis can be prevented or retarded by expressing a Bcl-2 encodingnucleic acid or functional equivalent thereof in viral-infected cells(see, for example, FIG. 3). Elevated levels of Bcl-2 inhibit theprogrammed cell death induced by an infecting viruses and result inprolonged survival of the infected cells. Bcl-2-containing viral vectorsthat appropriately target the infected cells can be used to specificallyintroduce and increase the Bcl-2 activity within the infected cells.Such vectors also can contain non-Bcl-2-encoding nucleic acids that areuseful for treating the virus-infected cells.

The invention provides a method of prolonging the in vivo survival oftransplanted cells for the treatment of a disease or pathologicalcondition. The method includes increasing the activity of Bcl-2 in apopulation of cells and transplanting the population of cells havingincreased Bcl-2 activity into a subject. Diseases or pathologicalconditions can include, for example, neurodegenerative diseases, cancerand virus-infected cells.

The transplantation of genetically modified cells that secrete proteins,hormones or neurotransmitters, for example, can be used to treat theabove diseases as well as many chronic, metabolic and inheriteddisorders such as diabetes and hemophilia. Employing the methodsdescribed herein, the in vivo survival of such diseased cells can beimproved by Bcl-2 gene transfer. An advantage of treating cells usingBcl-2 is that Bcl-2 is not oncogenic in most cells and, therefore, canbe used to “immortalize” cells that would be responsive to normal growthcontrol mechanisms in vivo.

Cell transplantation is now being explored for the treatment of certaindiseases, notably Parkinson's disease. For example, potential therapiesin animal models of Parkinson's disease have included celltransplantation of genetically modified fibroblasts, which produceL-DOPA in the vicinity of the substantia nigra. Although the results ofthese experiments have been encouraging, the survival time of thetransplanted cells is limited and, therefore, results in only atemporary and minor improvement of the condition.

Transplantation of fetal brain cells, which contain precursors of thedopaminergic neurons, has also been examined as a potential treatmentfor Parkinson's disease. In animal models and in patients with thisdisease, fetal brain cell transplantations have resulted in thetemporary reduction of motor abnormalities. Furthermore, it appears thatthe implanted fetal dopaminergic neurons form synapses with surroundinghost neurons. However, the transplantation of fetal brain cells is againlimited due, for example, to the limited survival time of the implantedneuronal precursors.

In the specific case of Parkinson's disease, intervention by increasingthe activity of Bcl-2 can improve the in vitro and in vivo survival offetal and adult dopaminergic neurons, their precursors anddopamine-secreting fibroblasts and, thus, can provide a more effectivetreatment of this disease. Likewise, improved in vivo survival ofessentially any cell type to be transplanted will improve the treatmentof that disease. For example, neuronal cells or their precursors can beused for the treatment of other neurodegenerative diseases such asAlzheimer's disease and glutamate-induced neuronal cell death byenhancing the in viva survival of cells using Bcl-2.

Specific examples of cell types other than neuronal cells includehepatocytes for the treatment of liver failure, β cells for thetreatment of insulin-dependent diabetes and skin cells for the treatmentof burns. Additionally, Bcl-2 expression can be used to enhance survivalof transplanted cells for cosmetic treatments. One example of such acosmetic purpose is for the treatment of alopecia, the medical term forbaldness. Moreover, viral vectors can be employed using the methods thattarget Bcl-2 expression to the cells of the hair follicle or Bcl-2transfer vehicles can be applied topically to the scalp, resulting in anovel genetic treatment for hair loss.

Cells to be transplanted for the treatment of a particular disease canbe genetically modified in vitro so as to increase the activity ofBcl-2. Such methods are known within the art and are essentially thesame as those described above, except that Bcl-2 expression is firstachieved within the cells in vitro. Bcl-2-expressing vectors can beconstructed using recombinant DNA techniques and can utilize, forexample, viral vectors, viral genomes, plasmids, phagemids and the like(see, for example, FIG. 1). Such vectors can also encode one or morenon-Bcl-2 nucleotide sequences to facilitate the therapeutic function ofthe cells once they are transplanted.

Bcl-2-encoding vectors are introduced into recipient cells usingtransfection methods known to one skilled in the art. Such methodsinclude, for example, infection using viral vectors, lipofection,electroporation, particle bombardment and transfection. Detailedprocedures for these methods can be found in Sambrook et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989)and the references cited therein, which are incorporated herein byreference.

Following transfection, cells having increased levels of Bcl-2 activityare selected for use in transplantation treatment. The screeningprocedure will depend on the method by which the Bcl-2 activity isincreased. For example, if increased activity is accomplished throughelevated Bcl-2 protein levels, then a quantitative assay that determinesthe accumulated Bcl-2 protein level can be used. Such assays include,for example, immunoblot analysis, immunoprecipitation and ELISA. Suchmethods are known to one skilled in the art and can be found in Ausubelet al., Current Protocols in Molecular Biology (John Wiley and Sons,1989) or in Harlow et al., Antibodies: A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1988), both of which are incorporated herein byreference. Functional assays also can be employed such as the inhibitionof apoptotic DNA degradation or nuclear disintegration, such asdisclosed herein or known in the art (see Example VII).

Similar to the use of Bcl-2 for prolonged in vivo survival oftransplanted cells, the expansion of mammalian cells in culture forsubsequent transplantation or the expansion of cells for industrialscale production of proteins, metabolites or other clinically usefulfactors can benefit from the enhanced survival due to Bcl-2. Among thelimitations that plague these types of procedures are the dependence ofmany cell types on adherence to a solid surface. Moreover, many types ofcells die through apoptosis when their required growth factors run inshort supply or when toxic by-products accumulate in cultures of cellsgrown at high densities. Such problems can be overcome by increasing thelevels of Bcl-2 within the cultured cells.

For example, increased levels of Bcl-2 can allow anchorage-dependentcells to survive and grow in the absence of attachment to a solidsurface. Bcl-2 overexpression can also allow cells to grow to higherdensities when compared to cells expressing low or normal Bcl-2 levels.Moreover, a novel form of the Bcl-2 protein, Bcl2/P59S, is substantiallymore active than the wild-type protein in allowing higher density growthand preventing cell death due to the depletion of growth and survivalfactors in tissue culture medium (FIG. 2). All of these properties ofBcl-2 can be utilized for mass expansion of cells in culture.

The invention also provides a method of prolonging the in vivo survivalof transplanted cells for the treatment of a disease or pathologicalcondition by increasing the Bcl-2 activity of immune cells. The immunesystem consists of a variety of cell types that protect the body frominfectious organisms and continually monitor the body for the appearanceof abnormal cells such as cancer cells. Some immune cells have thecapacity to bind to and kill other cell types, particularly tumor cellsand virus-infected cells. One outcome of the killer cell response,besides target cell death, is the activation of apoptosis within thekiller cells, themselves. The physiological role of this self-inducedsuicide may be a negative feedback mechanism for controlling an immuneresponse. However, this regulatory mechanism of immune cell deathprevents immune responses from being of sufficient duration andintensity to effectively eliminate malignant or virus-infected cells.Tumor-specific and virus-specific immune cells can also die from lack ofsufficient growth factors such as interleukin-2 (Il-2) in vivo. Thisprocess can be arrested or prevented by Bcl-2 gene transfer. Thus, theaugmentation of immune cell survival due to increased Bcl-2 expressioncan result in more effective treatment of cancer and virus-induceddiseases.

Apoptotic death of immune cells can be inhibited by isolating thesecells or their precursors and modifying them to express elevated levelsof Bcl-2. The methods for modifying these cells are essentially the sameas those described above. The transplantation of the Bcl-2-expressingcells into a subject suspected of having a cancer or a viral infectionwill ensure a more prolonged and active immune response against thecondition. As an alternative to extracorporeal treatment,tissue-specific gene transfer and expression technology can be used tospecifically increase Bcl-2 gene expression in the killer cells in vivo.

Other non-Bcl-2 genes that augment the cell survival function of Bcl-2or enhance the killer activity of immune cells can be introduced intocells in combination with Bcl-2. A specific example of introducing asecond gene that enhances the effector activity of immune cells is thecoexpression of the Lck protein tyrosine kinase. When constitutivelyoverexpressed, for example, by mutation of the regulatory tyrosineresidue at amino acid position 505 to a phenylalanine (Y505F), Lckconfers T cell effector functions on the Y505F-expressing cell in theabsence of IL-2. Abrogation of the IL-2 requirement is clinicallyadvantageous because side effects due to treatment with animmunostimulatory drug such as IL-2 can be avoided. Nucleic acidsencoding proteins other than Lck can be coexpressed to enhance effectorfunctions of immune cells. Such nucleic acids include, for example, Lyn,Hck, Fyn, Yes, Atk, Fgr and Blk.

Raf-1, which encodes a serine-threonine protein kinase, is an example ofa non-Bcl-2 gene that can be administered in conjunction with Bcl-2 toenhance the action of Bcl-2 (see Example V and FIG. 6). Infection ofcells with DNA sequences encoding a mutant version of the Raf-1 kinasehaving constitutive, non-inducible kinase activity acts synergisticallywith Bcl-2 to prolong cell survival by blocking apoptotic cell death(see, for example, FIG. 6.A.). Thus, coexpression of non-Bcl-2 genes toaugment function of Bcl-2 or to enhance other desired functions providesa means of preventing or limiting virus infections and malignant cellgrowth.

The invention also provides a method for enhancing the sensitivity ofmalignant or virus-infected cells to therapy by decreasing the activityof Bcl-2 in the malignant or virus-infected cells. Decreased activitycan be accomplished by expressing an alternative form of Bcl-2 capableof forming a bound complex with Bcl-2, wherein the bound Bcl-2 isinactive, or with other proteins that interact with Bcl-2, thusinhibiting the normal function of Bcl-2.

Many types of malignant cells become resistant or refractory totreatment due to high endogenous levels of Bcl-2. These malignant cellshaving high levels of Bcl-2 include prostate, colorectal andnasopharyngeal cancers and lymphomas, leukemias and neuroblastoma.Similarly, virus-infected cells can be intrinsically resistant totreatment because of endogenous Bcl-2 expression. In contrast to thepreviously described methods for increasing the Bcl-2 activity, suchdiseases can be effectively treated by utilizing the opposite approach,i.e., inhibiting Bcl-2 activity. Suppression of Bcl-2 function can beemployed alone or in combination with conventional therapies to providea more effective means of decreasing the resistance of malignant orvirus-infected cells to killing by chemotherapeutic drugs andirradiation.

Bcl-2 expression can be inhibited, for example, by targeting and/orexpression vectors that produce the alternative form of the Bcl-2protein, Bcl-2β. When expressed in conjunction with a normal Bcl-2 gene,Bcl-2β binds to the cellular proteins with which Bcl-2 normallyinteracts and prevents Bcl-2 function, probably through a competitionmechanism. The inhibition of a wild-type function through thecoexpression of a variant form of a gene product is known in the art asa dominant negative mutation (FIG. 7; see, also, Kolch et al., Nature349:426-428 (1991), which is incorporated herein by reference.

Bcl-2β arises through an alternative splicing mechanism and lacks thehydrophobic stretch of amino acids found in the normal Bcl-2 protein,the hydrophobic region being necessary for membrane insertion of Bcl-2and its function as a blocker of apoptosis. Other examples of mutantBcl-2 are proteins that have been genetically engineered to containdeletions within the region of amino acids 85-219 of the 239 amino acidBcl-2 protein. In malignant cells where Bcl-2 function has been markedlyreduced by dominant negative mutation, enhanced sensitivity to killingby a wide variety of chemotherapeutic drugs such as methotrexate,Adriamycin, Ara-C and dexamethasone can be observed.

In addition to inhibiting Bcl-2 activity to enhance the sensitivity ofmalignant or virus infected cells to therapy, other non-Bcl-2 geneproducts involved in the progression of the diseased state can beinhibited to increase the efficacy of treatment. For example, highlevels of Raf-1 kinase activity are associated with radioresistance oftumors. Gene transfer manipulations that cause an increase of decreaseof Raf-1 kinase activity can increase or decrease, respectively, thesensitivity of tumor cells to killing by chemotherapeutic drugs.Moreover, elevations in Raf-1 activity in the presence of increasedBcl-2 activity indicate that the combined use of reagents designed tointerfere with Raf-1 and Bcl-2 can act synergistically to render tumorcells more sensitive to killing by conventional chemotherapeutic drugsand irradiation (see Example V).

As an alternative to gene therapy and transfer approaches, chemicalcompounds that alter the activity of Bcl-2 can be used. The samerationale as described above for treating diseases or pathologicalconditions can be applied to these applications, except that thespecific compounds that alter the Bcl-2 activity are substituted inplace of recombinant methods. Thus, all the therapies describedpreviously using Bcl-2 gene transfer are equally applicable to the useof Bcl-2 specific compounds.

Novel Bcl-2 specific compounds can be obtained, for example, throughrational design or random drug-screening methods. All that is requiredis a method to accurately identify active compounds. Active compoundsinclude both those that increase Bcl-2 activity as well as those thatdecrease its activity. Thus, the determination of an activity willdepend on the desired outcome.

The invention provides a method to identify compounds that alter theprocess of apoptosis (see Example VII). This method includes treating anapoptotic cell extract with one or more compounds and selecting thecompound that alters the apoptotic process in the cell extract. Thus,the method allows the identification of active Bcl-2 specific compounds.The method consists of a cell free extract that faithfully reproducesthe apoptotic process. Briefly, when Xenopus egg extracts are mixed withsperm chromatin, the chromatin is assembled into a nucleus that issurrounded by a nuclear enveloped. These cell free nuclei undergodegeneration spontaneously with time or inducibly in the presence ofparticular drugs. The nuclear degeneration process is indicative of theprocess that occurs in cells dying by apoptosis. Addition of Bcl-2protein to the extracts prevents nuclear breakdown (see, for example,FIG. 9). Active Bcl-2 specific compounds can be identified bysubstituting the compound for Bcl-2 in the cell free extract. Anadvantage of the cell free extract method is that it can be automated bymonitoring, for example, the transport of radiolabeled orfluorescent-tagged peptides into nuclei; this transport process isprevented by nuclear breakdown.

The invention also provides a method to enhance monoclonal antibodyproduction by prolonging the in vitro survival of hybridoma precursorcells by increasing the activity of Bcl-2 in the precursor cells.Similar to the methods described above, Bcl-2 can enhance the survival,for example, of antibody producing cells. Such enhanced survival canincrease the efficiency of monoclonal antibody production by allowingthe generation of a greater number of successful fusions.

Precursor hybridoma cells, such as myeloma fusion partners andantibody-producing B cells, can be modified using methods describedherein to elevate the expression of Bcl-2. Bcl-2 expression vectors canbe introduced in vitro into the myeloma cells using the disclosedmethods. Since the antibody-producing B cells are isolated from animmunized animal, increasing Bcl-2 expression in these cells can beaccomplished by immunizing transgenic animals expressing a Bcl-2encoding transgene. B cells taken from the spleen of such immunizedanimals will have enhanced survival characteristics compared to B cellsfrom normal animals. Thus, the invention also provides transgenic miceexpressing Bcl-2 as the transgene. Moreover, increased Bcl-2 activitycan also be used to immortalize human B cells for the production ofhuman monoclonal antibodies.

The following examples are intended to illustrate, but not limit theinvention.

EXAMPLE I

This Example shows that Bcl-2 expression results in an increased numberof viable 32D-3 cells in culture and allows the cells to remain viablefor longer than normally observed for this cell line.

32D-3 cells were stably transfected with expression plasmids containinga G418 antibiotic resistance gene and either a wild-type or mutant humanBcl-2 DNA sequence. The mutant Bcl-2 sequence encodes a variant Bcl-2protein having a serine substitution for a proline at amino acidposition 59 (BCL2/P59S). This amino acid substitution results in a highlevel of constitutive expression of BCL2/P59S.

Following selection of the cells for G418 resistance, cells were platedand their viability was determined. As shown in FIG. 2.A., the number ofviable control cells (Neo) reached a maximum about day 4-5, thendecreased. The number of viable cells expressing the wild-type Bcl-2protein (W.T.) also reached a maximum at about day 4-5, but the numberof viable cells did not decrease during the time examined. In contrast,the number of viable cells expressing the variant Bcl-2 protein (P59S)increased for 6 days to a level about two-fold greater than the controlcells and the cells expressing with the wild-type Bcl-2 protein. Asshown in FIG. 2.B., the number of viable cells is correlated to thelevel of DNA synthesis in the various populations of cells.

These results indicate that Bcl-2 causes an increase in the number ofviable cells in culture and allows the cells to remain viable for alonger period of time than normal.

EXAMPLE II

This Example illustrates the effectiveness of Bcl-2 at preventingSindbis virus-induced death of prostate cancer cells.

A rat prostate cancer cell line, AT-3, was infected with pZIP-BCL2 orpZIP-NEO (see FIG. 1.B.), then grown in the presence or absence ofSindbis virus. At various times after the addition of Sindbis virus,cells were collected and cell viability was determine using trypan blueexclusion.

As shown in FIG. 3, Sindbis virus infection resulted in the death ofessentially all of the AT-3 cells within 3 days (solid circles). Incontrast, AT-3 cells that expressed Bcl-2 were protected from Sindbisvirus-induced death (solid squares) and survived as if no Sindbis viruswas present (compare controls, open circles). These results show thatBcl-2 expression protects cells from death due to virus infection.

EXAMPLE III

This Example demonstrates the effectiveness of Bcl-2 for inhibition ofglutamate-induced apoptosis in cultured neuronal PC12 cells.

Glutamate is released from dying neurons in the brain during stroke andother conditions where neuronal cell survival is compromised including,perhaps, ALS. Neurons contain cell surface glutamate receptors thatappear to transduce poorly characterized signals leading to apoptoticcell death. In vitro studies have shown that glutamate treatment canresult in the death of cultured cortical neurons and PC12 cells.Glutamate-induced cell death induced is accompanied by internucleosomalDNA degradation and ultrastructural changes resulting from apoptosis.Bcl-2 expression protects neuronal cells from glutamate-induced celldeath.

The rat pheochromocytoma cell line, PC12, was stably infected withpZIP-BCL-2 or pZIP-NEO (see FIG. 1.A.), then treated with 30 mMglutamate for 24 hours. Following treatment, cell viability was assessedby MTT dye reduction assay as described in Tada et al., J. Immunol.Meth. 93:157-165 (1986). As shown in FIG. 4, Bcl-2 expression protectedthe PC12 cells from glutamate-induced death.

PC12 cells or other neuronal cell lines that are sensitive to treatmentwith glutamate can be infected with recombinant Bcl-2. High levelproduction of the human Bcl-2 protein is confirmed by immunoblotting.The resulting cell lines are cultured with various concentrations ofglutamate. After various times, cells are recovered from culture and DNAfragmentation is assessed by qualitative gel-electrophoresis assays andby quantitative biochemical methods.

A sample of cells are fixed and embedded for electron microscopicanalysis to determine whether ultrastructural changes indicative ofapoptosis occurs. The percentage of viable cells also is compared forBcl-2-expressing and control cells using a version of the MTT dyereduction assay or by recovering the cells from cultures using 20%pancreatin, resuspending them in medium containing propidium iodide anddetermining the percentage of viable cells by fluorescence microscopy orby fluorescent activated cell sorting.

A qualitative DNA fragmentation assay is performed using the gelelectrophoresis assay described in Sorenson et al., J. Natl. Canc. Inst.82: 749 (1990). In addition, quantitative DNA fragmentation assays areperformed. Briefly, cells are grown for 12-24 hours in medium containing1 μCi/ml 3H-thymidine to metabolically radiolabel the cellular DNA.Cells are washed three times and approximately 1×10⁶ are returned totheir usual culture media. The cells are incubated in the presence ofvarious concentrations of glutamate (0-50 mM). Four to 24 hours later,the cells are pelleted by centrifuged and the culture supernatant iscollected to determine the ³H-thymidine concentration by liquidscintillation counting (fraction 1).

The cells are resuspended in 5 mM Tris (pH 7.4), 10 mM EDTA, 0.5% TritonX100 and centrifuged at 13,000×g for 30 min. The radioactivity in theresulting supernatant (fraction 2), which contains low molecular weight(fragmented) DNA, and the pellet, which contains intact high molecularweight DNA (fraction 3), is determined. The percentage of DNAfragmentation is calculated by summing the counts per minute (cpms) ofradioactivity in fractions 1 and 2, and dividing by the total cpms ofradioactivity in all three fractions.

EXAMPLE IV

This Example demonstrates the similarity of action of viral and avianhomologs of human Bcl-2.

32D-3 cells were stably transfected with expression plasmids containingeither a hygromyin (HYG) or a G418 (NEO) antibiotic resistance gene andcDNA encoding either the human Bcl-2 protein, the chicken Bcl-2 proteinor the BHRF-1 protein from Epstein Barr Virus (viral homolog of Bcl-2).Production of the desired proteins or the corresponding mRNA wasverified by immunoblot or northern blot assays. Following transfection,cells were cultured without growth factor for various times and thenumber of viable cells was determined.

As shown in FIG. 5.A., the chicken Bcl-2 homolog was nearly as effectiveas the human Bcl-2 in prolonging cell viability in culture. Similarresults were observed for the viral BHRF-1 protein, which also increasedcell viability in the absence of growth factors (FIG. 5.B.). Cellproliferation studies demonstrated that the increase in viability wasnot, in fact, due to cell growth.

EXAMPLE V

This Example demonstrates that non-Bcl-2 proteins involved in cellsurvival act synergistically to prolong survival of 32D-3 cells invitro.

32D-3 cells were stably transfected with expression plasmids producingBcl-2 protein, activated Raf-1 protein or both Bcl-2 and Raf-1, thencultured under conditions of growth factor deprivation that causeapoptosis. At various times after removing the growth factor from themedium, cells were collected and viability was determined.

As shown in FIG. 6.A., the expression of Raf-1 alone had no effect oncell survival, whereas expression of Bcl-2 alone had a modest effect.Remarkably, expression of both Bcl-2 and Raf-1 proteins togetherresulted in synergistic prolongation of cell survival. Significantly, nodifference was observed in the level of DNA synthesis of the variouspopulations of cells (FIG. 6.B.), indicating that the combined effect ofBcl-2 and Raf-1 represents enhanced cell survival and not, for example,an ability to proliferate in the absence of growth factors.

Flow cytometry was used to confirm that the growth factor-deprived cellswere not proliferating. Cells were grown for 3 days in the presence orabsence of Il-3. Following incubation, cells were stained with propidiumiodide and the relative DNA content of the cells was determined. Asshown in FIG. 6.C., BCL2-expressing cells and BCL2/RAF-expressing cellsgrown in the presence of growth factor (panels a and c) exhibit similarpatterns of fluorescence intensity, as do the different cell lines grownin the absence of Il-2 (panels b and d). The reduction in the number ofcells having a higher DNA content is consistent with the greater numberof non-cycling G₀/G₁-phase cells present in the cells grown in theabsence of Il-3 (panels b and d).

EXAMPLE VI

This Example illustrates the effectiveness of a dominant-negative formof the Bcl-2 protein in accelerating 32D-3 cell death.

32D-3 cells were stably infected with retrovirus vectors containing agene conferring neomycin resistance alone or in combination with a DNAsequence encoding a 22 kDa form of the Bcl-2 protein, Bcl-2β (FIG.1.A.). Following infection, cells were cultured for 1 day without growthfactor and the number of viable cells was determined. As shown in FIG.7, expression of the dominant-negative Bcl-2β protein resulted in asignificant cell killing.

EXAMPLE VII

This Example shows that Bcl-2 inhibits apoptotic degradation of nucleiin a cell free system.

A cell-free assay for assessing Bcl-2 function was developed to rapidlyscreen compounds for ability to inhibit or enhance Bcl-2 proteinfunction. The assay utilizes in vitro self-assembled nuclei generated bymixing cell extracts derived from Xenopus laevis eggs with isolatedsperm chromatin. The nuclei assemble normally but, after about 2 hours,begin to disintegrate. The nuclei appeared contracted and had the sameappearance as isolated nuclei that had been treated briefly with anendonuclease. This phenomenon appears to represent apoptosis.Alternatively, isolated hepatocyte nuclei can be substituted for spermchromatin and mixed with the egg extracts, resulting in the samephenomenon.

To test the effect of Bcl-2 protein on nuclei, lysates were preparedfrom Sf9 insect cells infected with recombinant baculoviruses encodingeither Bcl-2 or an irrelevant protein such as β-galactosidase (β-gal) orthe Lck kinase (see Reed et al., Anal. Biochem. 205:70-76 (1992). Toconfirm that Bcl-2 was expressed in the insect cell lysates, Sf9 insectcells were infected with recombinant Bcl-2 baculovirus or recombinantLck baculovirus vectors. Infected cells were incubated for three days toallow expression of the gene products, then lysates were obtained andfractionated by SDS-PAGE electrophoresis. Following electrophoresis,proteins were transferred to membrane and probed with an antiserumraised against a synthetic peptide corresponding to part of thepredicted amino acid sequence of human Bcl-2. As shown in FIG. 8, Bcl-2protein was expressed in Sf9 cells infected with the Bcl-2 baculovirusvector (lanes 1 and 2) but not in uninfected cells (lane 6) or cellsinfected with a baculovirus expressing the Lck kinase (lane 5). Bcl-2protein comprised about 5-10% of the total extracted protein.

Sf9 cell lysates were added at a 1:90 dilution into the Xenopus extractsor to a final protein concentration of 10 μg/ml (see Newmeyer andWilson, Meth. Cell Biol. 36:607-634 (1991), which is incorporated hereinby reference). In extracts receiving the control Sf9 lysate, chromatincondensation began after about 2 hours (FIG. 9.A., upper panels, andFIG. 9.B.). Round, highly condensed bodies that stain brightly with theHoechst 33258 DNA dye formed and general contraction of the nuclei andfloating bits of condensed chromatin in the surrounding extract wereapparent (not shown). After about another 40 min of incubation, thenuclei had disintegrated completely (FIG. 9.B.) and a generallydispersed filamentous background of Hoechst fluorescence remained alongwith many small spherical or irregularly shaped aggregates of chromatin(not shown).

In contrast, when the Bcl-2-containing lysate was added, the nuclei wereprotected from this kind of degradation (FIG. 9.A., lower panels, andFIG. 9.B.). A considerable proportion of the nuclei remained intact andwere completely functional in nuclear transport of the fluorescentsubstrate, tetramethylrhodamine-labelled BSA conjugated to a syntheticpeptide corresponding to the nuclear localization signal of SV40T-antigen (TRITC-HSA-NLS). Note that in FIG. 9.A., the upper panelscontain two nuclei, only one of which is intact and accumulates thefluorescent transport substrate. The lower panels (Bcl-2 present) show atypical intact nucleus, which has accumulated the transport substrateand has grown considerably, acquiring new membrane by fusion of nuclearmembrane vesicles with the nuclear membrane. The DNA (FIG. 9. A., leftpanel) is concentrated in one part of the nucleus of the Bcl-2-treatedextract. This DNA localization roughly corresponds with the originalelongated shape of the sperm chromatin body and has a normal feathery,i.e., decondensed, appearance typical of the morphology of sperm nucleiassembled in standard, non-apoptotic Xenopus egg extracts.

Quantitation of the intact, transport-competent nuclei observed in atypical experiment revealed that, after 2 hours of incubation, about 50%of the nuclei in the Bcl-2-treated extracts were intact (in standard,non-apoptotic extracts the percentage of intact nuclei can range from50-90%), whereas only 8-10% of the control nuclei were intact, asmeasured by transport competence (FIG. 9.B.). As discussed above, nointact nuclei remained 40 min later in the control extracts. However,42% of the nuclei in extracts containing Bcl-2 remained intact andtransport-competent after two hours and forty min incubation. Thus, theBcl-2-containing lysates confer on nuclei in these extracts a protectionfrom degradation.

In order to show that the suppression of nuclear breakdown was due tothe presence of Bcl-2 protein, immunoprecipitation reactions wereperformed. Two different insect cell lysates were pre-incubated withanti-Bcl-2-specific antibodies or irrelevant antibodies prior toaddition to the Xenopus cell free apoptosis assay. As shown in FIG. 10,lysates incubated with anti-Bcl-2 antibodies lost the ability tosuppress nuclear degradation, whereas incubation with irrelevantantibodies had no effect on the ability of the lysates to suppressdegradation. Depletion of Bcl-2 protein following immunoprecipitation ofthe lysates with anti-Bcl-2 antibody was confirmed by western blotanalysis (not shown).

Additional results indicate that the action of Bcl-2 is not mediatedthrough changes in the efficiency of nuclear import. Measurement of thenuclear import activity in these same samples showed that the additionof Bcl-2-containing lysate resulted in no gross changes in the amount offluorescent transport substrate imported by nuclei in one hour.

As a result of the disclosure that nuclear lysis in the cell free systemis mediated by a pathway that can be blocked by Bcl-2, the system can beused to examine compounds generated, for example, by rational design orrandom synthesis, for the ability to alter apoptosis. Compounds thatexhibit the desired activity can be selected and used for treatingvarious diseases and pathological conditions.

EXAMPLE VIII

This example shows that Bcl-2 expression vectors can be used to prolongthe in vivo survival of genetically-engineered fibroblasts that secreteL-DOPA when transplanted into lesioned brains. A similar approach can beused when transplanting genetically modified myoblasts, neuroendocrineor other cell types. In addition, the vectors need not be directlyinjected but can be incorporated into biocompatible “capsules” that canbe implanted into the desired region of the brain.

Bcl-2 gene transfer is used for prolonging the survival ofintracranially implanted fibroblasts that are genetically engineered tosecrete L-DOPA. An immortal rat fibroblast line (208F) or primary ratskin fibroblasts stably infected with tyrosine hydroxylase-encodingrecombinant retroviruses has been grafted into the denervated striatumof rat brains. These transplanted cells reduce behavioral symptoms for 2and 10 weeks, respectively. The loss of a therapeutic effect after a fewweeks for 208F cells was a result of the loss of surviving transplantedcells. In this animal model of Parkinson's disease, the success of thetherapeutic approach has been hampered by the failure of transplantedfibroblasts to survive long-term in the brains of animals. Theexpression of Bcl-2 can circumvent this problem and prolong theintracranial survival of genetically engineered fibroblasts whentransplanted into the striatum of brains.

To generate fibroblasts coexpressing Bcl-2 and tyrosine hydroxylase(TH), Bcl-2 retroviruses are used to infect established and primary ratfibroblasts that previously stably infected with viruses expressing TH.Bcl-2/TH positive cells are selected and stereotactically introducedinto the brains of subjects having Parkinson's disease.

The methods for performing these injections in human patients aredescribed, for example, in Freed et al., New Engl. J. Med. 327:1549-1555 (1992) and Freed et al., Arch. Neurol. 47: 505-512 (1990).Briefly, before the operation, the caudate and putamen are visualized byCT scanning. Implantation is carried out through an ellipticalcraniectomy (3.5 by 1.5 cm) with the patient awake and sedated and underlocal anesthesia. The patient's head is mounted into a stereotacticframe and the CT scan is used to determine the coordinates forinjection. Six to nine needle passes are made on each side of the brainand 1.5-3 μl of cell suspension is deposited along 10 mm tracks in theputamen, caudate or both as the needle is slowly withdrawn.

An animal model that can be used to test the efficacy or optimize theprocedure is the use of rats, which have undergone 6-hydroxydopaminelesioning of their striatum neurons. For example, the in vivo lifespanof Bcl-2-expressing fibroblasts transplanted into the brains of suchrats is compared with the lifespan of fibroblasts infected with controlvirus. In this way, the appropriate number of cells required for aparticular Bcl-2-expressing cell line can be determined.

Experiments can be performed using the rat model. In this case,implantation is performed by mounting the anesthetized animal's head ina stereotactic frame and injecting 10 μl of cell suspension into twosites in the striatum using the coordinates AP=0.3 mm; ML 2.0 mm; DV=4.5mm, and AP=1.5 mm; ML=2.0 mm, DV=4.5 mm, as described in Paxinos andWatson, The Rat Brain in Stereotaxic Coordinates (Academic Press, 1982).At each site, 2.5 μl of cells are dispensed, then the syringe is raised1 mm and another 2.5 μl is administered at a rate of 1 μl/minute. Thesyringe is left in place for an additional 2 min to allow for diffusionof the cells, as described in Fisher et al., Neuron 6:371-380 (1991),which is incorporated herein by reference.

The retroviral vector used for stably infecting TH-positive fibroblastsexpresses both a Bcl-2 cDNA and the lacZ gene of E. coli. LacZ encodesthe β-gal, which is easily monitored by calorimetric assays. The salientfeatures of this vector include a Moloney sarcoma virus 5′-LTR followedby the Bcl-2 gene, an internal ribosome initiation site, lacZ and a 3′Moloney leukemia virus LTR. This retroviral construct results insignificantly higher titers of virus production than the popular pBAGvector (see Austin and Cepko, Development 110:713-732 (1990), which isincorporated herein by reference).

To produce the viral vectors, the above-described plasmid will betransfected by standard calcium phosphate precipitation into PE501, anecotrophic packaging cell line that has a lower incidence of spontaneousrecombination events than the more commonly used Psi-2 cells. As usedherein, the term “ecotrophic” means the viral vector has a limited hostrange, i.e., only infects rodent cell, as opposed to the term“amphotrophic,” which refers to a virus than infects cells from allspecies. The transiently produced virus is used to stably infect theamphotrophic cell line PA317 by the method of Miller et al., Som. CellMol. Gen. 12: 175-182 (1986), which is incorporated herein by reference.

After 2 passages in culture to allow for viral integration, stablyinfected cells are enriched by three rounds of fluorescence activatedcell sorting (FACS) using a fluorescent β-gal substrate that can be usedwith viable cells, Nolan et al., Proc. Natl. Acad. Sci. USA 85:2603-2607(1988), which is incorporated herein by reference. Sorted cells areseeded at low density into cultures or plated at limiting dilution intomicrotiter plates. Individual clones are randomly recovered, propagatedin culture and screened initially for virus expression based on relativelevels of Bcl-2 protein production as determined by immunoblot assayusing antibodies specific for human Bcl-2 protein.

Culture supernatants derived from promising clones are titered byinfection of NIH3T3 cells and scored for foci of blue cells in monolayercultures. This screening procedure uses a protocol that involves fixingthe cells in situ and staining with calorimetric substrates for β-gal,Lim and Chae, Biotechniques 7:576-579 (1989), which is incorporatedherein by reference. Clones exhibiting the highest virus production arethen analyzed by DNA blot to verify integrity of the integratedprovirus(es) and tested for contaminating helper virus by standardmethods (see, for example, Mann et al., Cell 33:153-159 (1983), which isincorporated herein by reference).

To obtain the Bcl-2/TH positive fibroblasts, the Bcl-2/β-gal vectorgenerated above is stably infected into TH positive rodent fibroblasts.The infected cells are enriched by flow cytometry and implanted into the6-hydroxydopamine lesioned brains of rats. Treatment is monitored byweekly evaluation for symptoms such as apomorphine- oramphetamine-induced rotations. Animals are sacrificed at various timesto determine the relative numbers of surviving cells by histochemicalanalysis of tissue sections stained blue by use of appropriatecalorimetric substrates of β-gal.

An alternative approach to the above-described β-gal-dependent selectionstrategy for optimizing transplantation procedures is to use otherretroviral vectors instead of the Bcl-2/β-gal vector. These othervectors contain, for example, an appropriate marker such as a hygromycinresistance gene, Gaken et al., Biotechniques 13:32-33 (1992), which isincorporated herein by reference. In this way, Bcl-2 cDNA is introducedinto G41-resistant, TH-expressing fibroblasts and the stably infectedcells are selected for growth in hygromycin. Where a colorimetric markergene is unavailable, the presence of Bcl-2 protein producing fibroblastsin the brains of rats is assessed, for example, by immunohistochemicalmeans using anti-Bcl-2 antibodies, which are specific for the exogenousform of the protein and useful for immunostaining formalin- or Bouin'sreagent-fixed, paraffin-embedded tissues.

EXAMPLE IX

This example shows the use of Bcl-2 expression vectors to prolong the invivo survival of transplanted fetal dopaminergic neurons.

Bcl-2 gene transfer is used for prolonging the in vitro survival ofdopaminergic neurons recovered from fetal neural tissues. ImmortalizedTH-expressing neuronal precursors were constructed using a v-mycretrovirus as the immortalizing oncogene. Fetal cells from themesencephalic flexure region of day 13 rat embryos were used because theneuronal cells that populate the substantia nigra and subsequentlyinnervated the striatum are thought to migrate through this area aroundgestational day 12 to 14, Marchlard and Poirier, Neuroscience 9:373-381(1983), which is incorporated herein by reference.

Cells were infected with a v-myc virus and placed into culture. By 12days post infection, all of the uninfected control cells were dead,whereas about half of the cells exposed to v-myc virus survived. Afterselection in G418, cells were cloned and propagated in culture for atleast 20 passages over 6 months. Three clones were selected at randomfor detailed characterization and one of them, termed MF13/H11, wasfound to contain TH.

As shown by immunohistochemical studies, MF13/H11 cells expressedtypical neuronal cell antigens such as the 150 kDa and 200 kDaneurofilament proteins, a neuron-specific adhesion protein, contactin,and a marker of the neural crest, ganglioside GD2. The cells did notexpress the glial cell and astrocyte markers GFAP and the proteoglycanNG2. The cells continue to divide in high-density conditions whenprovided with serum but atrophy and die when serum is withdrawn.MF13/H11 is the first immortalized dopaminergic neuronal precursor clonedescribed to date.

Although there is some reported success using v-myc ortemperature-sensitive versions of SV40 large T antigen forimmortalization of neuronal precursor cells and subsequentdifferentiation of these cells in vivo, the immortalized clones thatretain ability to differentiate are the exception rather than the rule.The TH-expressing MF13/H11 cells described above, for example, exhibitlittle ability to differentiate in vitro when stimulated with retinoicacid and other potent pharmacological inducers of neuronal celldifferentiation. Unlike v-myc, however, Bcl-2 has the capacity toimmortalize cells without affecting their ability to proliferate anddifferentiate.

To prolong the in vitro survival of fetal neuronal cells, the sameprocedure described above for the immortalization of these cells withv-myc is used except that Bcl-2 is used instead. Both an ecotrophic(PE501) and an amphotrophic cell line (PA317) are used to insure thatany preferences will be avoided.

Briefly, infections are performed by culturing the fetal neuronalprecursor cells for 1-2 days in tissue culture dishes coated withpoly-L-lysine or poly-L-ornithine. Such conditions eliminate glial cellsand favor the attachment of neuronal precursor cells. High-titer (>10⁶colony forming units/ml) virus-containing supernatants are collectedfrom fresh cultures of PA317 or PE501 cells and added to the fetal cellcultures for 3 to 4 hrs with 10 to 20 μg/ml polybrene or for 16-24 hrswith 4 μg/ml polybrene. Virus-containing media is then removed and thefetal cells are cultured for 1 to 2 days longer before adding theappropriate selection agent (G418, hygromycin or puromycin depending onwhether a neomycin phosphotransferase, hygromycin phosphotransferase orpuromycin-resistance gene, respectively, was included in the recombinantbcl-2 retrovirus). Continued culture in media containing theseantibiotics can be for as little as 1 day or for several months,depending on the goals of the experiment.

In some cases, no selection with antibiotics is performed. Here,selection of Bcl-2 infected cells is essentially performed using thesurvival-promoting functions of Bcl-2. Only Bcl-2 virus-infectedcultures will contain viable, proliferating cells since uninfectedmesencephalic cells typically are dead within two weeks. Alternatively,β-gal activity, Bcl-2 RNA or protein analysis can be used to screen forBcl-2-expressing neuronal precursor cells. The Bcl-2 stabletransformants are expanded and transplanted essentially as describedabove for the fibroblasts.

EXAMPLE X

This Example describes the production of transgenic mice expressingBcl-2.

The DNA construct used for the production of Bcl-2 transgenic mice wasconstructed as follows. A 4.3 kb fragment (λ1032−5/SH) containing aportion of the human Bcl-2 3′ untranslated region, t(14;18) breakpoint,heavy chain joining region gene segment and the IGH enhancer region wasexcised from the λ phage clone λ1032-5 (Tsujimoto et al., Science229:1390-1393 (1985), which is herein incorporated by reference) bydigestion using SstI and HindIII. The excised sequence was madeblunt-ended using DNA polymerase (Klenow fragment) and T4 polymerase andsubcloned into the HincII site of pSKII (Stratagene, La Jolla, Calif.).A 6.9 kb fragment (p18-21H/BH) containing two promoters and the firsttwo exons and a portion of the second intron of Bcl-2 was excised fromp18-21H by Hind III and partial BamHI digestion and subcloned into thepSKII/E_(μ) plasmid above, Tsujimoto et al., Proc. Natl. Acad. Sci. USA84: 1329-1331 (1987), which is herein incorporated by reference.Finally, a 4.4 kb HindIII fragment (p18-4/H) containing a portion of thesecond intron, third exon, 3′ untranslated region and polyadenylationsite of Bcl-2 was isolated from p18-4 and subcloned into the HindIIIsite located between the 5′ Bcl-2 and E_(μ) fragments described above.For microinjection, a 13-kb DNA fragment was isolated from the finalplasmid by BssHII digestion, gel purified and dialyzed against modifiedTE buffer (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA).

Transgenic mice were produced by injecting about 250-500 copies of theabove construct into the male pronucleus of (SWR/J×SJL/J) F₁ fertilizedeggs by standard methods known in the art. Integration of the transgenewas initially screened by PCR analysis of tail lysates using primersspecific for the t(14;18) major breakpoint region. Results wereconfirmed by DNA blot analysis of liver DNA isolated from F₁ progeny ofeach transgenic line. All backcrosses were with SWR/J mice. Relativelevels of Bcl-2 protein were measured by a two-stepimmunoprecipitation/immunoblot assay that uses antibodies specific forthe human Bcl-2 protein, as described in Reed et al., Canc. Res.51:6529-6538 (1991).

Although the invention has been described with reference to thedisclosed embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A method of prolonging the survival of a β-cell from a pancreaticislet comprising increasing the activity of Bcl-2 in said β-cell,wherein increasing the activity of Bcl-2 in said β-cell comprisesintroducing and expressing in said β-cell a nucleic acid moleculeencoding full length Bcl-2, Bcl-2/P59S or BHRF-1 and wherein introducingsaid nucleic acid molecule in said β-cell occurs in vitro, therebyprolonging the survival of said β-cell.
 2. The method of claim 1,wherein said nucleic acid molecule encodes human Bcl-2.
 3. The method ofclaim 1, wherein said nucleic acid molecule encodes Bcl-2/P59S.
 4. Themethod of claim 1, wherein said nucleic acid molecule encodes BHRF-1. 5.The method of claim 1, further comprising introducing into said β-cell anucleic acid encoding Raf-1.
 6. The method of claim 1, wherein saidβ-cells are human β-cells.
 7. A method of prolonging the survival oftransplanted β-cells, comprising increasing the activity of Bcl-2 in apopulation of β-cells from pancreatic islets and transplanting saidpopulation of β-cells having increased Bcl-2 activity into a subject,wherein increasing the activity of Bcl-2 in said population of β-cellscomprises introducing and expressing in said β-cells a nucleic acidmolecule encoding full length Bcl-2, Bcl-2/P59S or BHRF-1 and whereinintroducing said nucleic acid molecule in said β-cell occurs in vitro.8. The method of claim 7, wherein said subject is a human.
 9. The methodof claim 7, wherein said nucleic acid molecule encodes human Bcl-2. 10.The method of claim 7, wherein said nucleic acid molecule encodesBcl-2/P59S.
 11. The method of claim 7, wherein said nucleic acidmolecule encodes BHRF-1.
 12. The method of claim 7, further comprisingintroducing into said cells a nucleic acid encoding Raf-1.
 13. Themethod of claim 7, wherein said β-cells are human β-cells.
 14. Themethod of claim 7, wherein said subject is diabetic.
 15. The method ofclaim 7, wherein said subject is insulin-dependent.